Humanized and chimeric monoclonal antibodies to CD47

ABSTRACT

Humanized or chimeric anti-CD47 monoclonal antibodies are provided. The antibodies bind to and neutralize human CD47, and find use in various therapeutic methods. Preferred are non-activating antibodies. Embodiments of the invention include isolated antibodies and derivatives and fragments thereof, pharmaceutical formulations comprising one or more of the humanized or chimeric anti-CD47 monoclonal antibodies; and cell lines that produce these monoclonal antibodies. Also provided are amino acid sequences of the antibodies.

CROSS-REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 17/108,731, filed Dec. 1, 2020, which is a Continuation ofapplication Ser. No. 15/175,848, filed Jun. 7, 2016, which is aContinuation of application Ser. No. 14/656,431, Mar. 12, 2015, now U.S.Pat. No. 9,382,320, granted Jul. 5, 2016, which is a Divisional ofapplication Ser. No. 13/675,274, filed Nov. 13, 2012, now U.S. Pat. No.9,017,675, granted Apr. 28, 2015, which is a Continuation in Part of PCTApplication No. PCT/US2011/36535, filed May 13, 2011, which claimsbenefit of U.S. Provisional Patent Application No. 61/395,652 filed May14, 2010, which applications are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

Macrophages clear pathogens and damaged or aged cells from the bloodstream via phagocytosis. Cell-surface CD47 interacts with its receptoron macrophages, SIRPα, to inhibit phagocytosis of normal, healthy cells.CD47 is a broadly expressed transmembrane glycoprotein with a singleIg-like domain and five membrane spanning regions, which functions as acellular ligand for SIRPα with binding mediated through the NH₂-terminalV-like domain of SIRPα. SIRPα is expressed primarily on myeloid cells,including macrophages, granulocytes, myeloid dendritic cells (DCs), mastcells, and their precursors, including hematopoietic stem cells.

SIRPα inhibits the phagocytosis of host cells by macrophages, where theligation of SIRPα on macrophages by CD47 expressed on the host targetcell generates an inhibitory signal mediated by SHP-1 that negativelyregulates phagocytosis. SIRPα acts to detect signals provided by “self,”to negatively control innate immune effector function against thesecells.

In keeping with the role of CD47 to inhibit phagocytosis of normalcells, there is evidence that it is transiently upregulated onhematopoietic stem cells (HSCs) and progenitors just prior to and duringtheir migratory phase, and that the level of CD47 on these cellsdetermines the probability that they are engulfed in vivo.

CD47 is also constitutively upregulated on a number of cancers,including myeloid leukemias. Overexpression of CD47 on a myeloidleukemia line increases its pathogenicity by allowing it to evadephagocytosis. We conclude that CD47 upregulation is an importantmechanism that provides protection to normal HSCs duringinflammation-mediated mobilization, and that leukemic progenitors co-optthis ability in order to evade macrophage killing.

The present invention provides anti-CD47 antibodies having lowimmunogenicity in humans.

SUMMARY OF THE INVENTION

Compositions and methods are provided relating to humanized or chimericanti-CD47 monoclonal antibodies. The antibodies of the invention bind toand neutralize human CD47, and find use in various therapeutic methods.Preferred are non-activating antibodies. Embodiments of the inventioninclude isolated antibodies and derivatives and fragments thereof,pharmaceutical formulations comprising one or more of the humanized orchimeric anti-CD47 monoclonal antibodies; and cell lines that producethese monoclonal antibodies. Also provided are amino acid sequences ofthe antibodies.

Antibodies of interest include the provided humanized or chimericantibodies, and variants thereof. The monoclonal antibodies of theinvention find particular utility as reagents for the diagnosis andimmunotherapy of disease associated with CD47 in humans, particularly incancer therapy. An advantage of the monoclonal antibodies of theinvention derives from the humanization process. Thus, in vivo use ofthe monoclonal antibodies of the invention for immunotherapy greatlyreduces the problems of significant host immune response to theantibodies.

Various forms of the antibodies are contemplated herein. For example,the anti-CD47 antibody may be a full length chimeric or humanizedantibody, e.g. having a human immunoglobulin constant region of anyisotype, e.g. IgG1, IgG2a, IgG2b, IgG3, IgG4, IgA, etc. or an antibodyfragment, e.g. a F(ab′)₂ fragment, and F(ab) fragment, etc. Fragmentscomprising CDR regions are also of interest, e.g. for imaging purposes.Furthermore, the antibody may be labeled with a detectable label,immobilized on a solid phase and/or conjugated with a heterologouscompound. The antibody may also be provided as a bi-specific ormultispecific antibody reactive with a second antigen, particularlyincluding cancer antigens.

Diagnostic and therapeutic uses for the antibody are contemplated,particularly relating to the detection and elimination of undesirablecells expressing CD47. In one diagnostic application, the inventionprovides a method for determining the presence of CD47 expressing cancercells, comprising exposing a patient sample suspected of containing CD47expressing cancer cells to the anti-CD47 antibody and determiningbinding of the antibody to the sample. For this use, the inventionprovides a kit comprising the antibody and instructions for using theantibody.

The antibodies of the invention are particularly efficacious in thetreatment of disease, e.g. increasing the phagocytosis of CD47expressing cells. Treatment may be systemic or localized, e.g. deliveryby intratumoral injection, etc.

Embodiments of the invention include isolated antibodies and derivativesand fragments thereof that comprise at least one, usually at least 3 CDRsequences from a set, as provided herein, usually in combination withframework sequences from a human variable region or as an isolated CDRpeptide. In some embodiments an antibody comprises at least one lightchain comprising a set of 3 light chain CDR sequences provided hereinsituated in a variable region framework, which may be, withoutlimitation, a human or mouse variable region framework, and at least oneheavy chain comprising the set of 3 heavy chain CDR sequence providedherein situated in a variable region framework, which may be, withoutlimitation, a human or mouse variable region framework.

In other embodiments, the antibody comprises an amino acid sequencevariant of one or more of the CDRs of the provided antibodies, whichvariant comprises one or more amino acid insertion(s) within or adjacentto a CDR residue and/or deletion(s) within or adjacent to a CDR residueand/or substitution(s) of CDR residue(s) (with substitution(s) being thepreferred type of amino acid alteration for generating such variants).Such variants will normally having a binding affinity for human CD47 ofat least about 10⁻⁸ M and will bind to the same epitope as an antibodyhaving the amino acid sequence of those set forth herein. For example,the light chain CDR3 may be modified to mutate the de-amidation site.Various forms of the antibodies are contemplated herein. For example,the antibody may be a full length antibody, e.g. having a humanimmunoglobulin constant region of any isotype, e.g. IgG1, IgG2a, IgG2b,IgG3, IgG4, IgA, etc. or an antibody fragment, e.g. a F(ab′)₂ fragment,and F(ab) fragment, etc. Furthermore, the antibody may be labeled with adetectable label, immobilized on a solid phase and/or conjugated with aheterologous compound.

The invention further provides: isolated nucleic acid encoding theantibodies and variants thereof; a vector comprising that nucleic acid,optionally operably linked to control sequences recognized by a hostcell transformed with the vector; a host cell comprising that vector; aprocess for producing the antibody comprising culturing the host cell sothat the nucleic acid is expressed and, optionally, recovering theantibody from the host cell culture (e.g. from the host cell culturemedium). The invention also provides a composition comprising one ormore of the human anti-CD47 antibodies and a pharmaceutically acceptablecarrier or diluent. This composition for therapeutic use is sterile andmay be lyophilized, e.g. being provided as a pre-pack in a unit dosewith diluent and delivery device, e.g. inhaler, syringe, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. Amino acid sequences of B6H12 heavy chain variable region(A) (SEQ ID NO:1) and light chain variable region (B) (SEQ ID NO:2).Complementarity determining regions (CDR) are as indicated.

FIG. 2 . SDS-PAGE analysis of purified B6H12 proteins. Purified chimericand humanized B6H12 were analyzed by SDS-PAGE under non-reducingconditions. Molecular mass standards are indicated on the left.

FIG. 3A-3B. Competition of chimeric B6H12 antibody against mouse B6H12antibody for CD47 binding. A. Chimeric B6H12 competed against mouseB6H12 for binding to YB2/0 cells that had been stably transfected withhuman CD47 (YB2/0-CD47). A human IgG1 antibody was used as an isotypecontrol. B. Mouse B6H12 competed against chimeric B6H12 for binding tohuman CD47 expressed on transfected YB2/0 cells. A mouse IgG1 was usedas an isotype control.

FIG. 4A-4B. Nucleotide sequences for humanized B6H12 heavy chainvariable region (A) and (SEQ ID NO:9) and light chain variable region(B) (SEQ ID NO:10).

FIG. 5 . Comparison of the binding of chimeric and humanized B6H12antibodies to human CD47 by flow cytometry. YB2/0 cells stablytransfected with human CD47 were stained with chimeric B6H12, humanizedB6H12, or a human IgG1 isotype control antibody. Bound antibody wasdetected with PE-labeled secondary antibody.

FIG. 6 . Comparison of the binding of chimeric and humanized B6H12antibodies to human CD47 by ELISA. Soluble CD47 binding activity wasmeasured by ELISA as described in Materials and Methods. Bound antibodywas detected with goat anti-human kappa conjugated to HRP, and signalwas developed using OPT.

FIG. 7 . Chimeric and humanized B6H12 antibody-mediated phagocytosis.CFSE-labeled HL-60 cells were incubated with mouse bone marrow-derivedmacrophages in a 4:1 target to effector cell ratio. 2 hours later, themacrophages were imaged by fluorescence microscopy to detectphagocytosis. The phagocytic index (number of target cells ingested per100 macrophages) was determined for each condition in duplicate.Statistical comparison of each antibody to hIgG1 isotype control usingStudent's t-test showed all antibodies enabled a statisticallysignificant increase in phagocytosis (p-values: mouse B6H12 antibody:0.004; chimeric B6H12 antibody: 0.04; and humanized B6H12 antibody:0.003.)

FIG. 8A-8C. Amino acid alignment between humanized B6H12 VL and humanVK3-11 and JK1 JK1 (A) (SEQ ID NOs:12 and 34), and humanized B6H12 VHand human VH3-7 and JH4 (B) (SEQ ID NO:11 and 35). Number of differentamino acids of humanized B6H12 and human germline sequences in theframework and CDR regions of VH and VL are summarized in the table (C).

FIG. 9A-9B. Amino acid sequences of 5F9 heavy chain variable region (A)(SEQ ID NO:18) and light chain variable region (B) (SEQ ID NO:19).Complementarity determining regions (CDR) are as indicated.

FIG. 10A-10B. Amino acid sequences of 8B6 heavy chain variable region(A) (SEQ ID NO:26) and light chain variable region (B) (SEQ ID NO:27).Complementarity determining regions (CDR) are as indicated.

FIG. 11 . Comparison of the binding of chimeric 5F9 and 8B6 antibodiesto human CD47 by ELISA. Soluble CD47 binding activity was measured by anELISA assay as described previously. Bound antibody was detected withgoat anti-human kappa conjugated to HRP, and signal was developed usingOPT.

FIG. 12A-12B. Amino-acid sequence alignments of different versions ofhumanized 5F9 heavy chain variable regions (A) (from top to bottom SEQID NOs:36-40 and 18) and light chain variable regions (B) (from top tobottom SEQ ID NOs:41-44 and 19) with germline sequences. CDR regions areunderlined.

FIG. 13 . Comparison of the binding of humanized and chimeric 5F9antibodies to human CD47 by ELISA. Soluble CD47 binding activity wasmeasured by an ELISA assay as described previously. Bound antibody wasdetected with goat anti-human kappa conjugated to HRP, and signal wasdeveloped using OPT.

FIG. 14 . Phagocytosis induced by antibodies of 5F9 and 8B6. HL-60 cellswere used as target cells and incubated with human peripheralblood-derived macrophages in a 4:1 target to effector cell ratio. Eachcondition was done in duplicate.

FIG. 15 . ELISA binding to huCD47-mFc fusion with C3 antibody.

FIG. 16 . In vitro phagocyosis against HL-60 with C3 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to humanized monoclonal antibodies whichare specific for CD47. Also disclosed is a nucleic acid, and amino acidsequence of such antibodies. The antibodies find use in therapeutic anddiagnostic methods associated with CD47.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibodies” (Abs) and“immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains (Clothia et al., J. Mol.Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A.82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Variable region sequences of interest include the provided humanizedvariable region sequences for anti-CD47 antibodies: B6H12: SEQ ID NO:11(heavy chain), SEQ ID NO:12 (light chain); 5F9: SEQ ID NO:36 or 37 or 38(heavy chain); SEQ ID NO:39 or 40 or 41 (light chain); 8B6: SEQ ID NO:35(heavy chain), SEQ ID NO:36 (light chain); or C3: SEQ ID NO:53 (heavy)and SEQ ID NO:58 (light chain).

The CDR sequence sets of exemplary anti-CD47 heavy and light chainscombinations are set forth in the sequence listing, including B6H12: SEQID NO:3-8; 5F9: SEQ ID NO:20-25; 8B6: SEQ ID NO:28-33; and C3: SEQ IDNO:61-63 heavy and SEQ ID NO:64-66 light. In some embodiments the CDRsequences for a particularly heavy and light chain combination as setforth in B6H12, 5F9, 8B6 or C3 will be maintained in a combination, i.e.a humanized antibody will comprise both B6H12 heavy chain CDR sequencesand B6H12 heavy chain CDR sequences; or both 5F9 heavy chain CDRsequences and 5F9 heavy chain CDR sequences, or 8B6 heavy chain CDRsequences and 8B6 heavy chain CDR sequences, or both C3 heavy chain CDRsequences and C3 light chain CDR sequences.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species(scFv), one heavy- and one light-chain variable domain can be covalentlylinked by a flexible peptide linker such that the light and heavy chainscan associate in a “dimeric” structure analogous to that in a two-chainFv species. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety and (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety; andmultispecific or multivalent structures formed from antibody fragments.In an antibody fragment comprising one or more heavy chains, the heavychain(s) can contain any constant domain sequence (e.g. CH1 in the IgGisotype) found in a non-Fc region of an intact antibody, and/or cancontain any hinge region sequence found in an intact antibody, and/orcan contain a leucine zipper sequence fused to or situated in the hingeregion sequence or the constant domain sequence of the heavy chain(s).

Unless specifically indicated to the contrary, the term “conjugate” asdescribed and claimed herein is defined as a heterogeneous moleculeformed by the covalent attachment of one or more antibody fragment(s) toone or more polymer molecule(s), wherein the heterogeneous molecule iswater soluble, i.e. soluble in physiological fluids such as blood, andwherein the heterogeneous molecule is free of any structured aggregate.A conjugate of interest is PEG. In the context of the foregoingdefinition, the term “structured aggregate” refers to (1) any aggregateof molecules in aqueous solution having a spheroid or spheroid shellstructure, such that the heterogeneous molecule is not in a micelle orother emulsion structure, and is not anchored to a lipid bilayer,vesicle or liposome; and (2) any aggregate of molecules in solid orinsolubilized form, such as a chromatography bead matrix, that does notrelease the heterogeneous molecule into solution upon contact with anaqueous phase. Accordingly, the term “conjugate” as defined hereinencompasses the aforementioned heterogeneous molecule in a precipitate,sediment, bioerodible matrix or other solid capable of releasing theheterogeneous molecule into aqueous solution upon hydration of thesolid.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Each mAb is directedagainst a single determinant on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they canbe synthesized by hybridoma culture, uncontaminated by otherimmunoglobulins. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made in an immortalized B cell or hybridoma thereof, ormay be made by recombinant DNA methods.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-CD47 antibody with a constant domain (e.g. “humanized”antibodies), or a light chain with a heavy chain, or a chain from onespecies with a chain from another species, or fusions with heterologousproteins, regardless of species of origin or immunoglobulin class orsubclass designation, as well as antibody fragments (e.g., Fab, F(ab′)₂,and Fv), so long as they exhibit the desired biological activity.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 75% by weight of antibody as determined bythe Lowry method, and most preferably more than 80%, 90% or 99% byweight, or (2) to homogeneity by SDS-PAGE under reducing or nonreducingconditions using Coomassie blue or, preferably, silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. Ordinarily, however, isolated antibody will be prepared by atleast one purification step.

The term “epitope tagged” when used herein refers to an anti-CD47antibody fused to an “epitope tag”. The epitope tag polypeptide hasenough residues to provide an epitope against which an antibody can bemade, yet is short enough such that it does not interfere with activityof the CD47 antibody. The epitope tag preferably is sufficiently uniqueso that the antibody specific for the epitope does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least 6 amino acid residues and usually between about 8-50 aminoacid residues (preferably between about 9-30 residues). Examples includethe c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto(Evan et al., Mol. Cell. Biol. 5(12):3610-3616 (1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,Protein Engineering 3(6):547-553 (1990)).

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody.The label may itself be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

Polypeptides

In one aspect, the present invention is directed to humanized orchimeric monoclonal antibodies that are specifically reactive with andneutralize CD47, and cell lines that produce such antibodies. Variableregions of exemplary antibodies are provided. Antibodies of interestinclude these provided combinations, as well as fusions of the variableregions to appropriate constant regions or fragments of constantregions, e.g. to generate F(ab)′ antibodies. Variable regions ofinterest include at least one CDR sequence of the provided anti-CD47antibody, where a CDR may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or moreamino acids. Alternatively, antibodies of interest include a variableregion as set forth in the provided antibodies, or pairs of variableregions sequences as set forth herein.

In some embodiments a polypeptide of interest has a contiguous sequenceof at least about 10 amino acids, at least about 15 amino acids, atleast about 20 amino acids, at least about 25 amino acids, at leastabout 30 amino acids, up to the complete provided variable region.Polypeptides of interest also include variable regions sequences thatdiffer by up to one, up to two, up to 3, up to 4, up to 5, up to 6 ormore amino acids as compared to the amino acids sequence set forthherein. In other embodiments a polypeptide of interest is at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 99% identical to the amino acid sequence set forth herein.

In addition to Fabs, smaller antibody fragments and epitope-bindingpeptides having binding specificity for at least one epitope of CD47 arealso contemplated by the present invention and can also be used in themethods of the invention. For example, single chain antibodies can beconstructed according to the method of U.S. Pat. No. 4,946,778 to Ladneret al, which is incorporated herein by reference in its entirety. Singlechain antibodies comprise the variable regions of the light and heavychains joined by a flexible linker moiety. Yet smaller is the antibodyfragment known as the single domain antibody, which comprises an isolateVH single domain. Techniques for obtaining a single domain antibody withat least some of the binding specificity of the intact antibody fromwhich they are derived are known in the art. For instance, Ward, et al.in “Binding Activities of a Repertoire of Single Immunoglobulin VariableDomains Secreted from Escherichia coli,” Nature 341: 644-646, disclose amethod for screening to obtain an antibody heavy chain variable region(H single domain antibody) with sufficient affinity for its targetepitope to bind thereto in isolate form.

The invention also provides isolated nucleic acids encoding thehumanized or chimeric anti-CD47 antibodies, vectors and host cellscomprising the nucleic acid, and recombinant techniques for theproduction of the antibody. Nucleic acids of interest may be at leastabout 80% identical to the provided nucleic acid sequences, at leastabout 85%, at least about 90%, at least about 95%, at least about 99%,or identical. In some embodiments a contiguous nucleotide sequence asset forth in any one of the provided coding sequences of at least about20 nt., at least about 25 nt, at least about 50 nt., at least about 75nt, at least about 100 nt, and up to the complete provided sequence maybe used. Such contiguous sequences may encode a CDR sequence, or mayencode a complete variable region. As is known in the art, a variableregion sequence may be fused to any appropriate constant regionsequence.

For recombinant production of the antibody, the nucleic acid encoding itis inserted into a replicable vector for further cloning (amplificationof the DNA) or for expression. DNA encoding the monoclonal antibody isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of the antibody). Many vectorsare available. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The anti-CD47 antibody of this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologousor homologous polypeptide, which include a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide, an immunoglobulin constant regionsequence, and the like. A heterologous signal sequence selectedpreferably may be one that is recognized and processed (i.e., cleaved bya signal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the native antibody signal sequence, thesignal sequence is substituted by a prokaryotic signal sequenceselected.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

Suitable host cells for cloning or expressing the DNA are theprokaryote, yeast, or higher eukaryote cells. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1.982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). Host cells are transformed with the above-describedexpression or cloning vectors for anti-CD47 antibody production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). Thematrix to which the affinity ligand is attached is most often agarose,but other matrices are available. Mechanically stable matrices such ascontrolled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. Where the antibody comprises a CH₃ domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Methods of Use

The humanized or chimeric monoclonal antibodies of the invention can beused in the modulation of phagocytosis, including the methods set forthin International Application US2009/000319, herein specificallyincorporated by reference in its entirety. For example, antibodycompositions may be administered to increase phagocytosis of cancercells expressing CD47.

The humanized or chimeric monoclonal antibodies of the invention can beused in vitro and in vivo to monitor the course of CD47 disease therapy.Thus, for example, by measuring the increase or decrease in the numberof cells expressing CD47, particularly cancer cells expressing CD47, itcan be determined whether a particular therapeutic regimen aimed atameliorating disease is effective.

The monoclonal antibodies of the invention may be used in vitro inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. In addition, the monoclonal antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize monoclonal antibodies of theinvention are flow cytometry, e.g. FACS, MACS, immunohistochemistry,competitive and non-competitive immunoassays in either a direct orindirect format; and the like. Detection of the antigens using themonoclonal antibodies of the invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of CD47 expressingcells. Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art, which find use as tracers in therapeuticmethods, for use in diagnostic methods, and the like. For diagnosticpurposes a label may be covalently or non-covalently attached to anantibody of the invention or a fragment thereof, including fragmentsconsisting or comprising of CDR sequences. Examples of the types oflabels which can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, and bio-luminescent compounds. Those of ordinary skill in theart will know of other suitable labels for binding to the monoclonalantibodies of the invention, or will be able to ascertain such, usingroutine experimentation. Furthermore, the binding of these labels to themonoclonal antibodies of the invention can be done using standardtechniques common to those of ordinary skill in the art.

In some embodiments the antibody or a fragment thereof is attached to ananoparticle, e.g. for use in imaging. Useful nanoparticles are thoseknown in the art, for example including without limitation,Raman-silica-gold-nanoparticle (R—Si—Au—NP). The R—Si—Au-NPs consist ofa Raman organic molecule, with a narrow-band spectral signature,adsorbed onto a gold core. Because the Raman organic molecule can bechanged, each nanoparticles can carry its own signature, therebyallowing multiple nanoparticles to be independently detectedsimultaneously by multiplexing. The entire nanoparticle is encapsulatedin a silica shell to hold the Raman organic molecule on the goldnanocore. Optional polyethylene glycol (PEG)-ylation of R—Si—Au-NPsincreases their bioavailability and provides functional “handles” forattaching targeting moieties (see Thakor et al (2011) Sci Transl Med.3(79):79ra33; Jokerst et al. (2011) Small. 7(5):625-33; Gao et al.(2011) Biomaterials. 32(8):2141-8; each herein specifically incorporatedby reference).

For purposes of the invention, CD47 may be detected by the monoclonalantibodies of the invention when present in biological fluids and ontissues, in vivo or in vitro. Any sample containing a detectable amountof CD47 can be used. A sample can be a liquid such as urine, saliva,cerebrospinal fluid, blood, serum and the like, or a solid or semi-solidsuch as tissues, feces, and the like, or, alternatively, a solid tissuesuch as those commonly used in histological diagnosis.

Another labeling technique which may result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific anti-hapten antibodies.

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

Therapeutic formulations comprising one or more antibodies of theinvention are prepared for storage by mixing the antibody having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. The antibody composition will beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the antibody to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent the CD47associated disease.

The therapeutic dose may be at least about 0.01 μg/kg body weight, atleast about 0.05 μg/kg body weight; at least about 0.1 μg/kg bodyweight, at least about 0.5 μg/kg body weight, at least about 1 μg/kgbody weight, at least about 2.5 μg/kg body weight, at least about 5μg/kg body weight, and not more than about 100 μg/kg body weight. Itwill be understood by one of skill in the art that such guidelines willbe adjusted for the molecular weight of the active agent, e.g. in theuse of antibody fragments, or in the use of antibody conjugates. Thedosage may also be varied for localized administration, e.g. intranasal,inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v.,and the like.

The antibody need not be, but is optionally formulated with one or moreagents that potentiate activity, or that otherwise increase thetherapeutic effect. These are generally used in the same dosages andwith administration routes as used hereinbefore or about from 1 to 99%of the heretofore employed dosages.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The anti-CD47 antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Inaddition, the anti-CD47 antibody is suitably administered by pulseinfusion, particularly with declining doses of the antibody.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the anti-CD47 antibody.The label on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Example 1 Cloning and Generation of Monoclonal Antibodies DirectedAgainst Human CD47

We describe here the cloning, construction and expression of monoclonalantibodies directed against human CD47. From a mouse hybridoma cell linesecreting B6H12, a functional blocking antibody directed against humanCD47, total RNA was prepared and converted into cDNA using Ig-specificoligonucleotides. Heavy and light chain encoding cDNA fragments wereisolated and sequenced. Chimeric genes were then constructed by linkingthe murine V region cDNA fragments to human immunoglobulin constantregions. By competitive FACS analysis, chimeric B6H12 inhibited thebinding of native mouse B6H12 antibody to CD47, demonstrating that thechimeric and mouse B6H12 antibodies recognize the same epitope of CD47.Furthermore, we designed and constructed a humanized B6H12 antibody byCDR grafting. The humanized B6H12 antibody showed comparable CD47binding to that of chimeric B6H12. Both chimeric and humanized B6H12antibodies enable phagocytosis of cancer cells in vitro. We anticipatethat the chimeric and humanized antibodies will be less immunogenic thanthe native mouse antibody when administered to human patients as part ofanti-cancer therapy.

We have identified and validated leukemia stem cell-preferentialexpression of CD47 using antigen specific monoclonal antibodies. CD47 isa widely expressed transmembrane protein; however, we found that CD47was more highly expressed on AML LSC than their normal counterparts, andthat increased CD47 expression predicted worse overall survival in threeindependent cohorts of adult AML patients. CD47 serves as the ligand forsignal regulatory protein alpha (SIRPα), which is expressed onphagocytic cells including macrophages and dendritic cells, that whenactivated initiates a signal transduction cascade resulting ininhibition of phagocytosis. When we used a blocking monoclonal antibodydirected against CD47, it preferentially enabled phagocytosis of AML LSCand inhibited their engraftment in vivo. Furthermore, treatment of humanAML LSC-engrafted mice with anti-CD47 antibody depleted AML and targetedAML LSC. These results establish a rationale for anti-CD47 monoclonalantibodies as monotherapy or combination therapy for AML and othercancers.

Here we report the isolation, synthesis, and generation of a human IgG1chimeric monoclonal antibody-derived from B6H12, and a humanized B6H12antibody engineered by CDR grafting. We describe the construction ofchimeric and humanized immunoglobulin genes composed of the cDNAsencoding the variable regions of heavy and light chains fused to humanγ1 and K constant regions, respectively. Introduction of these genesinto mammalian cells resulted in production of functional chimeric andhumanized antibodies able to bind human CD47 and cause phagocytosis oftarget cells.

Materials and Methods

Antibody V cloning and sequencing. The cloning strategy used hereinvolved the initial isolation of RNA from hybridoma cells (Qiagen), andpreparation of cDNA. cDNA sequences encoding the heavy and light chainvariable regions of the B6H12 monoclonal antibody were obtained using 5′RACE-PCR techniques (Clontech) and were sequenced using standard DNAsequencing techniques.

Construction of B6H12/hIgG1 chimeric antibody. In order to construct theheavy and light chain variable regions of B6H12 in an expression vector,the following primers were used:

VH sense primer, (SEQ ID NO:45)5′ CAGACCCGTCGACATGAACTTCGGGCTCAGCTTGATTTTCCTT 3′  VH antisense primer,(SEQ ID NO:46) 5′ GCCCTTGGTGCTAGCTGAGGAGACGGTGACTGAGGTTCCTTGACC 3′ VL sense primer, (SEQ ID NO:47)5′ CGCCATCACAGATCTATGGTGTCCACTTCTCAGCTCCTTGGACTT 3′ VL antisense primer, (SEQ ID NO:48)5′ TGCAGCCACCGTACGTTTGATTTCCAGCTTGGTGCCTCCACCGAA 3′.PCR was then performed using cloned pfu DNA polymerase (Invitrogen).These PCR products were cut by SalI/NheI for the VH and BgIII/BsiwI forthe VL and ligated into an expression vector encoding human gamma 1 andkappa constant regions that was digested by SalI/NheI or BgIII/BsiwI,respectively. All constructs were sequenced to confirm sequencefidelity.

Molecular Modeling. Humanization of mouse anti-CD47 B6H12 antibody wasperformed by installing CDR residues from the mouse antibody into humangermline framework (FR) sequences. Briefly, mouse B6H12 was humanized byjudicious recruitment of corresponding CDR residues and a few FRresidues into the human sequences. Differences between mouse B6H12 andthe human FR residues were individually modeled to investigate theirpossible influence on CDR conformation. Humanized VH and VL genes weresynthesized by McLab (South San Francisco, Calif.).

Cell transfection and stable cell line establishment. Stable cell linesexpressing chimeric or humanized B6H12 were established by transfectionof the expression construct into CHOS cells using DMRIEC transfectionreagent (Invitrogen) according to the manufacturer's instructions. Threedays later, transfected cells were selected under 500 ug/ml G418. Stableclones were isolated by limited dilution in 96-well plates. To screenG418-resistant clones for their ability to secrete antibody,supernatants of the transfected cells were tested by ELISA. Briefly,96-well plates (Nunc, Roskilde, Denmark) were coated with 1 ug/ml goatanti-human Fc gamma antibody in PBS for 16 h at 4° C. After blocking for1 h with 0.4% BSA in PBS at room temperature, isolated supernatants wereadded in 1/3 sequential dilutions, and incubated for 1 h at roomtemperature. Plates were subsequently washed three times and incubatedwith HRP-conjugated goat anti-human kappa-specific antibody for 1 h atroom temperature. After washing, plates were developed with OPT. Thereaction was stopped with 2M H₂SO₄, and OD was measured at 520 nM.Positive clones were further expanded and expression was confirmed byELISA.

Antibody purification and characterization. The culture supernatant wasapplied to protein G Sepharose columns. The column was washed withphosphate-buffered saline (PBS) pH 8.0, and protein was then eluted witheluting buffer (glycine pH 2.0). The eluted fractions were collected intubes containing neutralizing buffer (2M Tris-HCl, pH 8.0) to adjust thepH to approximately 7.0. Finally, purified samples were dialyzed againstphosphate-buffered saline (PBS). Purity of the eluted antibody fractionwas analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) on 10% gels under reducing or non-reducingconditions. Bands were visualized by Coomassie brilliant blue staining.

Binding specificity by ELISA. Microtiter plates were coated with 100 μlpurified human CD47-Fc fusion protein at 1.0 μg/ml in PBS, and thenblocked with 100 μl of 0.4% BSA in PBS. Dilutions of the B6H12 chimericor humanized antibody were added to each well and incubated for 1 hourat room temperature. A known murine anti-CD47 antibody was used as apositive control, and human IgG1 was used as an isotype control. Theplates were washed with PBS/Tween and then incubated with a goatanti-human kappa-specific secondary reagent conjugated to horseradishperoxidase for 1 hour at room temperature. After washing, the plateswere developed with OPT substrate, and analyzed at OD of 520 nm.

Binding specificity by FACS. YB2/0 cells that had been stablytransfected with human CD47 were incubated with various amounts ofchimeric B6H12, humanized B6H12, or a human IgG1 isotype controlantibody on ice for 1 hr. The cells were washed three times with FACSbuffer (PBS containing 0.5% BSA and 0.05% NaN₃). PE labeled goatanti-human antibody was added as a secondary antibody, and the sampleswere incubated on ice for another 1 hour. Samples were washed andanalyzed using a FACSAria (Becton-Dickinson, San Jose, Calif., USA).

Competitive binding assay by FACS. Binding inhibition of the chimericB6H12 antibody to human CD47 by the mouse antibody B6H12 or an isotypecontrol antibody was measured using FACS. CD47-transfected YB2/0 cellswere harvested and washed twice with FACS buffer (PBS containing 0.5%BSA and 0.05% NaN₃). Then chimeric B6H12 was added to the cells at afinal concentration of 1 ug/ml with various amounts of mouse B6H12antibody or an isotype control antibody and incubated for 1 hour on ice.Similarly, binding inhibition of the mouse B6H12 antibody to human CD47was measured by adding various amounts of the chimeric B6H12 or anisotype control antibody. The samples were washed with FACS buffer, PElabeled goat anti-human or anti-mouse antibody was added, and thesamples were incubated on ice for another 1 hour. Samples were washedand analyzed using a FACSAria (Becton-Dickinson, San Jose, Calif., USA).

In vitro phagocytosis assays. HL-60 cells were CFSE-labeled andincubated with mouse bone marrow-derived macrophages in the presence of10 μg/ml IgG1 isotype control, mouse B6H12, chimeric B6H12, or humanizedB6H12 antibody for 2 hr. Cells were then analyzed by fluorescencemicroscopy to determine the phagocytic index (number of cells ingestedper 100 macrophages). Statistical analysis using Student's t-test wasperformed with GraphPad Prism.

Results

Cloning of mouse B6H12 variable regions. Using universal antibodyprimers, clones encoding heavy and light chain variable regions weresuccessfully isolated from an anti-CD47 B6H12 hybridoma. Multiple clonesof each V gene product were sequenced to monitor PCR-induced errors. TheVH and VL sequences are shown in FIGS. 1A and B, respectively. DNAsequence analysis for products demonstrated that the heavy chain ofB6H12 uses a V segment of the Igh-v7183 VH5 family, and that the lightchain belongs to the IGKV23 subgroup. The heavy chain variable regioncomprises CDR1, CDR2, and CDR3 sequences; and the light chain variableregion comprises CDR1, CDR2, and CDR3 sequences (FIG. 1 ).

Production and characterization of B6H12 chimeric antibodies. Toconstruct the expression vector for chimeric B6H12 antibody, the heavychain variable region of B6H12 that includes its native signal peptidesequence at the NH₂ terminus was fused to the constant region of thehuman γ1 heavy chain and then cloned into a mammalian expression vector.Similarly, the light chain variable region of B6H12 that includes itsnative signal peptide sequence at the NH₂ terminus was fused to theconstant region of human κ light chain and introduced into the vectorencoding the B6H12 heavy chain. The resulting single expression vectorwas then transfected into mammalian cells. Expressed chimeric B6H12antibody was purified and examined by SDS-PAGE analysis. As expected,one single band with a molecular mass of ˜150 kDa was observed undernonreducing conditions (FIG. 2 ). After reduction with2-mercaptoethanol, two bands appeared at 50 kDa and 25 kDa,corresponding to heavy and light chains, respectively. These resultsindicate that chimeric heavy and light chain peptides produced in thetransfectant were assembled to form the native IgG molecule.

To demonstrate that B6H12 variable regions cloned from the mousehybridoma retain antigen binding activity similar to the original mouseB6H12 antibody, a competition binding assay between chimeric and mouseB6H12 was conducted by flow cytometry. Human CD47 was stably transfectedinto YB2/0 cells and the expression of CD47 was confirmed by flowcytometry. As shown in FIG. 3A, chimeric B6H12 competed out mouse B6H12for CD47 binding in a dose-dependent manner, while a human IgG1 isotypecontrol antibody had no impact on mouse B6H12 binding. Similarly, mouseB6H12 antibody inhibited chimeric B6H12 antibody for CD47 binding (FIG.3B). This suggests that the chimeric B6H12 antibody recognizes the sameepitope of CD47 as the native mouse B6H12 antibody.

Design and analysis of humanized B6H12 antibody. In order to selecthuman antibody frameworks (FR) to be used as templates for CDR-grafting,mouse B6H12 VL and VH regions were compared with those of human germlinesequences. The FRs of the mouse B6H12 VL region were found to have thehighest homology with the human VK3 subgroup, suggesting that a memberof subgroup III might be the best selection. The FRs of the mouse B6H12VH region exhibited the highest homology with the human VH-3 subgroup.The FRs from human VH-3-7 and VK3-11 were ultimately selected as thestarting point for designing humanized B6H12. Residues in the FRsidentical to the mouse sequences were retained and non-identicalresidues were either retained or substituted based on molecularmodeling. The humanized B6H12 was transfected and purified as describedabove. SDS-PAGE analysis showed one single band with a molecular mass of˜150 kDa under non-reducing conditions (FIG. 2 ), and two bands appearedat 50 kDa and 25 kDa under reducing conditions. The sequences are shownin FIG. 4 .

Next, the ability of humanized B6H12 to recognize human CD47 wasexamined. Human CD47-transfected YB2/0 cells, which have been shown toexpress membrane-bound CD47, were used for flow cytometry analysis.Humanized B6H12 bound CD47 expressed on the cell surface, and thebinding activity was equivalent to the chimeric B6H12 antibody (FIG. 5). No binding was detected with B6H12 antibodies when untransfectedYB2/0 cells were used. Similar results were also obtained whendetermining soluble CD47 binding by ELISA. In this assay, humanizedB6H12 showed comparable binding activity to chimeric B6H12 antibody(FIG. 6 ).

Enabling of phagocytosis by chimeric and humanized B6H12 antibodies.Mouse B6H12 antibody is known to block the interaction between CD47 andSIRPα, an inhibitory receptor expressed on macrophages, and therebyenable phagocytosis of the CD47-expressing cells. To examine the abilityof chimeric and humanized B6H12 antibodies to enable phagocytosis, weconducted an in vitro phagocytosis assay. CFSE-labeled HL-60 cells wereincubated with mouse bone marrow-derived macrophages for 2 hr inpresence of control or B6H12 native, chimeric, or humanized antibody.Phagocytosis was assessed by counting the number of ingestedCFSE-labeled HL-60 target cells within the mouse macrophages visualizedby fluorescence microscopy. As shown in FIG. 7 , both chimeric andhumanized B6H12 efficiently enabled phagocytosis, at levels comparableto that of the native mouse B6H12 antibody. In contrast, an isotypecontrol antibody did not trigger macrophage-mediated phagocytosis. Theseresults demonstrate that chimeric and humanized B6H12 are able tofunction in a similar manner as the native mouse B6H12 antibody.

Thus far, antibodies generated against human CD47 have been mouseantibodies. A major disadvantage of using a mouse antibody in thetreatment of human patients is the development of a human anti-mouseresponse (HAMA) in the patient. Accordingly, the need exists forimproved therapeutic antibodies against CD47 that are less immunogenic.In the present study, we constructed and expressed chimeric andhumanized antibodies engineered from variable regions of a mouseanti-human CD47 mAb (B6H12), which were fused to human immunoglobulinconstant regions. SDS-PAGE analysis revealed that both the chimeric andhumanized B6H12 antibodies are expressed as native IgG proteins composedof two pairs of heavy and light chains. Chimeric and mouse B6H12antibodies competed each other for antigen binding (FIG. 3 ), indicatingthat chimeric B6H12 antibody retains the antigen binding of the mouseantibody and recognizes the same antigen epitope. Furthermore, humanizedB6H12 antibody binds to both soluble and membrane-bound CD47equivalently to the chimeric antibody (FIGS. 5 and 6 ). Chimeric andhumanized B6H12 also showed efficient ability to enable phagocytosis ascompared to the original mouse B6H12 antibody (FIG. 7 ). These resultssuggest that our engineered antibodies form functionally active IgGs.

Notably, in B6H12 antibody humanization, we utilized human VH-3-7 andVK3-11 as the basis for our design. However, mouse B6H12 also showedsequence homology to other family members in the human VH-3 and VK3subgroups and to other variable domains outside these two subgroups. Itis possible other frameworks may work just as well.

Antibodies exhibit four main effector functions: antibody-dependentcellular cytotoxicity (ADCC), phagocytosis, complement-dependentcytotoxicity (CDC), and half-life/clearance rate. Each of these effectorfunctions is mediated through interaction with a specific set ofreceptors and cell types: ADCC and phagocytosis through interaction ofcell-bound mAbs with Fc gamma receptors (FcγR), CDC through interactionof cell-bound mAbs with the series of soluble blood proteins thatconstitute the complement system (e.g. Clq, C3, C4, etc.), andhalf-life/clearance rate through binding of antibodies to the neonatalFc receptor (FcRn). Activating antibodies, typically of the human IgG1subclass, are dependent on an activating Fc-domain. Monoclonalantibodies that function by blocking a ligand-receptor interaction,however, can function without utilizing effector mechanisms. In thesecases, effector functions may be disadvantageous as they may contributeto unwanted cytotoxicity. Unwanted agonism through crosslinking byFcR-expressing cells could trigger inappropriate activation ofFcR-expressing cells and subsequent cytokine storm and associated toxiceffects. Therefore, proper choice of IgG subclass or use of an IgGengineered to abrogate effector function is required. As we reportedpreviously, murine B6H12 functioned as a blocking antibody, and a B6H12F(ab)′2 fragment showed similar efficacy to the full length murine B6H12in in vitro phagocytosis assays. Thus, development of non-activatingB6H12 monoclonal antibodies with fewer side effects is beneficial.

Many strategies have been reported to engineer non-activatingantibodies. The use of antibody-based fragments lacking an Fc-domainprovides the simplest way to avoid Fc-dependent effector mechanisms.From a manufacturing point of view, antibody-based fragments representan attractive strategy as high yields are routinely obtained inwell-characterized and cost-effective lower eukaryotic and prokaryoticexpression systems. Recombinant antibody technologies have mademonovalent (e.g. Fab, scFv, nanobodies, and dAbs), bivalent (e.g.F(ab′)₂, diabodies, and minibodies) and multivalent (e.g. triabodies andpentabodies) formats available. These approaches have already resultedin FDA-approved therapeutics, and several others are undergoing clinicalevaluation, illustrating the confidence in this approach. However,removal of the Fc-domain will dramatically change the pharmacokineticproperties of antibody-based fragments and make antibody purificationless convenient. Without an Fc-domain, renal clearance is thepredominant mechanism influencing serum half-life and antibody-basedfragments smaller than ˜50-70 kDa are subject to this route ofelimination. Increasing the apparent molecular size of small antibodyfragments, for instance through linkage to polyethylene glycol (PEG) andhuman serum albumin (HSA), represents an alternative strategy toincrease circulation time and to improve their pharmacokineticproperties.

Combinations of therapeutic antibodies are also increasingly being usedthat may bring the additional benefit of targeting multiple epitopes orantigens. Combinations may be more effective against disease targetsthat are commonly heterogeneous and may thereby limit resistance orescape. We have demonstrated synergy and cure with B6H12 in combinationwith rituximab in human NHL xenotransplantation models. Our findingssuggest that combination therapy with B6H12 is a promising new treatmentmodality for NHL. Meanwhile, over the last few years, the concept ofbispecific antibody (BsAb)-mediated tumor cell killing has been studiedextensively both in preclinical models and clinical trials. BsAbs sharetwo different antigen-recognizing moieties within one molecule. Based onour data, B6H12 will synergize with additional FcR-engaging antibodiesto eliminate target cells. This synergy may be recapitulated in B6H12BsAbs reactive with CD47 on one hand and an additional surface antigenon a tumor target cell on the other. Such a reagent may focus immuneeffector functions towards the target cells.

In summary, we have developed therapeutic antibodies based on the mousemonoclonal antibody B6H12 directed against human CD47, by using methodsto create a mouse/human chimeric antibody and a humanized antibody. Thechimeric and humanized B6H12 antibodies retain the ability tospecifically bind CD47 and are able to induce phagocytosis in vitro.These antibodies may be less immunogenic, and thus are more suitable aspotential clinical therapeutics.

Example 2 Cloning of Mouse 5F9 and 8B6 Variable Regions

Using universal antibody primers, DNA fragments encoding heavy and lightchain variable regions were successfully cloned from anti-CD47hybridomas 5F9 and 8B6. Multiple clones of each V gene product weresequenced to monitor PCR-induced errors. The VH and VL sequences of 5F9are shown in FIGS. 9A and 9B, respectively. DNA sequence analysis forproducts demonstrated that the heavy chain of 5F9 uses a V segment ofthe Igh-VJ558 VH1 family, and that the light chain belongs to the IGKV1subgroup. The VH and VL sequences of 8B6 are shown in FIGS. 10A and 10B,respectively. DNA sequence analysis for products demonstrated that theheavy chain of 8B6 uses a V segment of the Igh-VJ558 VH1 family, andthat the light chain belongs to the IGKV23 subgroup. The heavy chainvariable regions comprise CDR1, CDR2, and CDR3 sequences; and the lightchain variable regions comprise CDR1, CDR2, and CDR3 sequences (FIGS. 9and 10 ).

CD47 binding activities of chimeric 5F9 and 8B6. Chimeric 5F9 and 8B6were constructed and expressed. Purified antibodies were analyzed bySDS-PAGE, demonstrating that native IgG antibodies were formed for both.Then, binding activities of chimeric 5F9 and 8B6 were tested using ELISAby coating human CD47 soluble protein in the 96-well plates. As shown inFIG. 11 , both chimeric 5F9 and 8B6 bound the antigen at comparablelevels to that of chimeric B6H12 antibody.

Antibody humanization and characterization of 5F9. In order to selecthuman antibody framework regions (FR) to be used as templates forCDR-grafting, the mouse 5F9 VL and VH regions were compared with thoseof human germline sequences. The FRs of the mouse 5F9 VL region werefound to have the highest homology with IGKV2 subgroup. The FRs of themouse 5F9 VH region exhibited the highest homology with human VH-1subgroup. Identical residues in the FRs were retained and non-identicalresidues were either retained or substituted based on molecularmodeling. Three versions of each humanized VH and VL were designed.Sequence alignments of each version together with human germlinesequences are indicated in FIG. 12 .

Different versions of humanized 5F9 heavy and light chains weretransfected in combinations, yielding different versions of humanized5F9. Then, the ability of humanized 5F9 antibodies to recognize CD47 wasexamined. As shown in FIG. 13 , both version 1 and version 2 ofhumanized 5F9 bound well to soluble CD47. Isotype control antibody didnot show any binding activity. These results demonstrate that humanized5F9 retained binding capability to the human CD47 antigen.

Phagocytosis induced by 5F9 and 8B6 antibodies. B6H12 antibody is knownto block the interaction between CD47 and SIRPα that is expressed onmacrophages and in turn to activate macrophages for phagocytic response.To examine 5F9 and 8B6 antibodies ability to induce phagocytosis, weincubated the antibodies with human peripheral blood-derived macrophagesand HL-60 target cells for 2 hr, and assessed phagocytosis by countingthe number of ingested CFSE-labeled HL-60 cells under a microscope. Asshown in FIG. 14 , both mouse and chimeric 5F9 were able to elicitphagocytosis efficiently, as did chimeric 8B6. Moreover, humanized 5F9antibodies also displayed effective phagocytic activity. In contrast,isotype control antibodies did not trigger macrophage-mediatedphagocytosis. These results demonstrate that 5F9 and 8B6 are able tofunction in a similar manner as B6H12 antibody.

What is claimed is:
 1. A method of detecting the presence of CD47 in abiological sample or tissue, the method comprising: contacting saidsample or tissues with an antibody that specifically binds to humanCD47, wherein said light chain comprises each of the CDR sequences setforth in SEQ ID NO:23-25; SEQ ID NO:31-33, or SEQ ID NO:64-66; andwherein said heavy chain comprises each of the CDR sequences set forthin SEQ ID NO:20-22 or SEQ ID NO:28-30 or SEQ ID NO:61-63.
 2. The methodof claim 1, wherein said light chain comprises the amino acid sequenceset forth in SEQ ID NO:19, 27, 41, 42 or
 43. 3. The method of claim 1,wherein said heavy chain comprises the amino acid sequence set forth inSEQ ID NO:18, 26, 36, 37 or
 38. 4. A method of detecting the presence ofCD47 in a biological sample or tissue, the method comprising: contactingsaid sample or tissues with an antibody that specifically binds to humanCD47, wherein said antibody comprises a heavy chain having each of theCDR sequences set forth in SEQ ID NO:20-22 and a light chain having eachof the CDR sequences set forth in SEQ ID NO:23-25; or a heavy chainhaving each of the CDR sequences set forth in SEQ ID NO:28-30 and alight chain having each of the CDR sequences set forth in SEQ IDNO:31-33; or a heavy chain having each of the CDR sequences set forth inSEQ ID NO:61-63 and a light chain having each of the CDR sequences setforth in SEQ ID NO:64-66.
 5. The method of claim 4, wherein the antibodyis a humanized monoclonal antibody.
 6. The method of claim 4, whereinthe antibody is a chimeric antibody.
 7. The method of claim 1, whereinthe antibody variable light (VL) region comprises an amino acid sequenceselected from the group consisting of the SEQ ID NO:41, SEQ ID NO: 42and SEQ ID NO: 43; and variable heavy (VH) region comprises an aminoacid sequence selected from the group consisting of the SEQ ID NO:36,SEQ ID NO: 37 and SEQ ID NO:
 38. 8. The method of claim 1, wherein theantibody variable light (VL) region comprises amino acid sequence SEQ IDNO:19; and variable heavy (VH) region comprises amino acid sequence SEQID NO:18.
 9. The method of claim 1, wherein the antibody variable light(VL) region comprises amino acid sequence SEQ ID NO:27; and variableheavy (VH) region comprises amino acid sequence SEQ ID NO:26.